The molecular mechanisms that lead to the expression of newly synthesized mediators of inflammation are the central focus of our studies. We utilize a mast cell model, RBL-2H3, which is capable of producing both inflammatory and immunoregulatory cytokines in response to the activation of the high affinity receptor for IgE (FcepsilonRI). In particular the steps involved in the signaling pathway, beginning with IgE-antigen activation of the high affinity receptor for IgE and resulting in the induction of cytokine genes, are being explored. In the past year we have focused principally on the in vivo interactions of tyrosine and serine/threonine kinases, characterization of multi-component signaling complexes and induction of transcription factors known to be critical for gene expression. Three principal areas of investigation were developed: 1) Studies on the regulation of molecular interactions between two kinase families (Src and PKC) and analysis of the functional effects of these interactions. 2) Regulation of transcription factor synthesis in response to aggregation of the FcepsilonRI. 3) Analysis of a role for the hematopoeitic cell specific protein-Vav in mast cell function. Our new results show: 1) The calcium independent PKC-delta associates with p60 Src and p53/56 Lyn. The association with the latter requires the aggregation of the FcepsilonRI while association with p60 Src is independent of receptor activation. Tyrosine phosphorylation of PKC-delta is required for its interaction with p53/56 Lyn suggesting that the SH2 domain of Lyn interacts with PKC-delta. We have found that tyrosine phosphorylation of PKC-delta regulates its substrate selectively. This study suggests that crosstalk between kinase families may serve as a novel intracellular mechanism for regulation of substrate recognition. 2) In collaborative studies we have determined that the transcription factor USF-2, which is thought to function in proliferative responses, is translationally regulated and aggregation of the FcepsilonRI results in increased USF-2 protein. The increased USF-2 translation is dependent on the activation of PKC-beta. These results provide the first evidence for a PKC-dependent mechanism of translational regulation of gene expression. 3) Aggregation of the FcepsilonRI results in association of Vav with this receptor. We have found that Vav, a protein thought to play a role in MAP kinase activation and therefore gene transcription, exists as a multicomponent complex in association with the adaptor molecule Grb2, the serine/threonine kinase Raf-1 and the p42 MAP Kinase. Functional analysis of the Raf-1 activity in this complex revealed that Vav-associated Raf-1 is constitutively active. These studies provide evidence of a link between receptor and MAP kinase activation.